EPA-600/3-77-108
September 1977
Ecological Research Series
A FLOW-THROUGH TESTING PROCEDURE WITH
DUCKWEED, (lemna minor L)
imental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal spe-
cies, and materials. Problems are assessed for their long- and short-term influ-
ences. Investigations include formation, transport, and pathway studies to deter-
mine the fate of pollutants and their effects. This work provides the technical basis
for setting standards to minimize undesirable changes in living organisms in the
aquatic, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service. Springfield, Virginia 22161.
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EPA-600/3-77-108
September 1977
A FLOW-THROUGH TESTING PROCEDURE WITH DUCKWEED (LEMNA MINOR L.)
by
Charles T. Walbridge
Environmental Research Laboratory-Duluth
Duluth, Minnesota 55804
ENVIRONMENTAL RESEARCH LABORATORY-DULUTH
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
DULUTH, MINNESOTA 55804
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DISCLAIMER
This report has been reviewed by the Environmental Research Laboratory-
Duluth, U.S. Environmental Protection Agency, and approved for publication.
Mention of trade names or commercial products does not constitute endorsement
or recommendation for use.
ii
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FOREWORD
Our nation's fresh waters are vital for all animals and plants, yet our
diverse uses of water — for recreation, food, energy, transportation, and
industry — physically and chemically alter lakes, rivers, and streams. Such
alterations threaten terrestrial organisms, as well as those living in water.
The Environmental Research Laboratory in Duluth, Minnesota, develops methods,
conducts laboratory and field studies, and extrapolates research findings
—to determine how physical and chemical pollution affects aquatic
life,
—to assess the effects of ecosystems on pollutants,
—to predict effects of pollutants on large lakes through use of
models, and
—to measure bioaccumulation of pollutants in aquatic organisms that
are consumed by other animals, including man.
This report describes a flow-through testing procedure for duckweed.
It identifies the variables that have to be controlled and the ranges to
which they should be confined. Copper sulfate is used as an example toxicant.
Donald I. Mount, Ph.D.
Director
Environmental Research Laboratory
Duluth, Minnesota
iii
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ABSTRACT
Lemna minor is one of the smallest flowering plants. Because of its
floating habit, ease of culture, and small size it is well adapted for
laboratory investigations. Procedures for flow-through tests were developed.
Testing procedures were developed with this apparatus. By using the techniques
described here, the effects of nutrients or toxicants, singly or in combination,
can be determined in several concentrations with several replicates of each.
Responses which can be measured include changes in growth rate, changes in
death rate, changes in timing of division of colonies, color changes, changes
in variability, and modification of the flowering response. Emphasis here is
on changes in growth rate determined either by daily frond counts or by final
frond numbers.
IV
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CONTENTS
Foreword iii
Abstract iv
Acknowledgments vi
1. Introduction 1
2. Conclusions and Recommendations 3
3. Methods 4
Physical methods 4
Biological methods /
4. Results 9
5. Discussion 13
Recommended Methods 15
References 17
Bibliography 19
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ACKNOWLEDGMENTS
The author gratefully acknowledges the technical guidance and editorial
assistance of Dr. Richard L. Anderson of the Environmental Research Laboratory-
Duluth.
vi
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SECTION 1
INTRODUCTION
Lemna minor L., lesser duckweed, is common throughout the world. In
fact, it is absent only from the polar regions and a few parts of the tropics.
Lemna minor is a small floating plant consisting of a single leaf-like frond
that is flat, more or less oval in outline, and 1.5 - 4 mm long. A single
root is attached about a third of the way from the narrow end of the frond.
The root, which is terminated by a prominent calyptra, or root cap, may be as
long as 10 cm. Vegetative reproduction is accomplished by the growth of
daughter fronds alternately from a pair of reproductive pouches at the sides
of the root-bearing end of the frond. Flowering is rare, but the flowers also
arise from the reproductive pockets. A Lemna colony is made up of several
generations of daughter fronds still attached to the parent. In its natural
environment ]j. minor is eaten heavily by some wildfowl, beaver, and muskrat.
It serves as physical support for epiphytic organisms, which are in turn
eaten by aquatic crustaceans, molluscs, annelids, and insect larvae. For fish
L_. minor provides food, shelter, and shade, though it tends to shade out
other aquatic plants that might produce food more efficiently (Muenscher 1944,
Sculthorpe 1967).
The Lemnaceae in general have a number of characteristics that make them
useful laboratory organisms, such as ease in obtaining clonal material,
simple methods of axenic culture, and small size. A consequence of this
combination of properties is that a large background of research has been
developed for this species.
Static tests are conducted in a non-renewed solution of the material under
investigation. Also, the plants may be transferred into fresh solutions during
the test. Several authors describe applicable techniques. Fromm (1946, 1951,
1960) used greenhouse lighting and 20 plants per flask with four flasks per
concentration. Mitchell et al. (1958) recommended sterilization of the
plants and culture medium for some uses. Further, they control light (700 -
1000 ft-C for 12 hrs per day) and temperature (24°- 27°C). Blackman (1952)
controlled light and temperature. He did not use sterile solutions or
sterilize the plants, but instead changed solutions every 48 hrs to keep
contamination down as well as to keep the pH and the chemical concentrations
within reasonable ranges. Fekete ££ aJ^- (1976) used a bioassay procedure to
evaluate the release of available phosphorus from pond sediments. Here again,
greenhouse lighting was used.
Advantages of the static and renewal procedures lie in the small quantities
of solution required and in the simplicity of the experimental design; no
delivery system is required. The major limitation of a static experiment is
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that the concentrations of active materials in the experimental container may
change over the test interval through degradation, precipitation, etc.
Flow-through and recirculating systems of varying degrees of sophistication
have been designed. These systems range from single open chambers, through
which the solution moves by a siphon arrangement (Mendiola 1919), to complex
aseptic systems in which the area of a raft of plants can be kept constant so
that the effects of crowding can be eliminated (Erismann and Finger 1968) or
systems in which samples may be taken automatically without violating aspesis
(Strasser 1971).
Ashby ^t al. (1928).designed a recirculating system that was not aseptic.
Eichorn and Augsten (1969) cultured Wolffia arrhiza in a chemostat under
continuous illumination.
The Lemnaceae have also been used in.systems for testing the effects of
various air pollutants on plants. Todd et_ a^. (1956) tested the effects of
air containing ozone and ozonated hexane. Feder and Sullivan (1969) studied
the reduction of frond multiplication and floral production by ozone. Scharer
et al. (1975) showed that Lemna minor could metabolize sulfur dioxide from
the air if it was made available in sublethal concentrations.
The cultivation and testing of Lemna minor have produced a number of
ways to quantify responses. These depend on such things as frond death,
premature division of colonies, alteration of flowering response, and dry
weight changes. At least two methods of visually evaluating frond condition,
are available. One is to assign degree-of-damage numbers to the fronds, e.g.,
from zero (no.damage) to five (complete loss of green color) (Parker 1965).
The second visual method involves frond counts, which have been shown to be a
reliable measure of growth response in the Lemnaceae. These counts compare
well with dry weight changes and area changes. Ashby and Oxley (1935) and
Fromm (1960) have used this technique.
The genus Lemna is unusual in that plants are not damaged by what might
be considered excessive day lenghts, including continuous illumination with
no dark period. The continuous growth of Lemna in a 24-h photoperiod is
useful in reducing the duration of the tests being performed (Clark 1925,
Landolt 1957).
The light intensities that can be used by Lemna minor range from 1,600 to
17,000 lux, but there is no appreciable increase in growth above about 8,000
lux. (Ashby and Oxley 1935). The lower end of this range is easily attainable
in laboratory situations.
The procedures described above were not designed with simplicity in mind,
so this work was undertaken to provide a fast, simple, and•inexpensive technique
for evaluating stimulatory or inhibitory effects of pollutants on a typical
aquatic plant.
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SECTION 2
CONCLUSIONS AND RECOMMENDATIONS
The lesser duckweed (Lemna minor L.) is a suitable higher plant for
testing in a flow-through system. The testing method developed in this
study should be useful for evaluating effects of toxicants, enrichments, and
otherwise altered conditions on higher aquatic plants. A procedure for
using Lemna minor as a test organism is recommended.
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SECTION 3
METHODS
Physical Methods
Lake Superior water, temperature-controlled but otherwise unmodified,
was delivered at regular intervals by use of a delivery chamber that
periodically emptied itself by a self-starting siphon (Hero of Alexandria,
ca. 62 AD). The periodic flow of water operated a chemical-metering apparatus
designed for highly water-soluble materials (Mount and Brungs 1967) (Figure 1).
This apparatus added a solution of plant nutrients to the feedwater of a
proportional diluter (Mount and Brungs 1967). This diluter was provided with
a mechanical syringe injector which added copper sulfate when this chemical was
used as a toxicant (DeFoe 1975) . The diluter delivered the resulting series of
concentrations through glass delivery tubes into battery jars. The jars had a
capacity of 1.4 1 each. The delivery tubes were wrapped in black polyethylene
film to reduce growth of attached algae on the inner surfaces. Toxicant was
added by an injector system designed to add microliter quantities to the water
to operate as the diluter cycled.
The battery jars drained through a notch cut 4 cm down from the top edge.
A silicone-based glass and ceramic adhesive was used to fasten stainless steel
bolting cloth (screen) across this opening. This allowed the water to drain
out, but prevented any loss of experimental plants. The screen used here was
105 mesh with 0.165-am openings.
Four separate battery jars were used for each concentration of the material
being tested. Individual colonies of duckweed were kept separated and centered
in the jars by seven-compartment floats (Figure 2). The separator consisted of
seven rings, 1-cm sections of 2—cm glass tubing; six of these were glued in a
hexagon around the seventh. This separator was glued between and supported by
two air-filled 4-, dram glass vials with plastic caps. The float was kept
centered with a hook made of bent stainless steel wire which was hung over the
edge of the jar. The separation of the colonies made it possible to monitor
each one individually. An index mark on one float showed the starting point
so that data for each colony could be recorded separately and consistently
(Figure 2).
Continuous illumination was provided by fluorescent tubes, half of which
were Durotest Optima and the remainder Gro Lux, Wide Spectrum. The distance
from the fluorescent tubes to the frond level was 20-25 cm. This provided a
light intensity of 2,700 lux; the standard deviation (s) was 2.50 lux.
Temperatures in the test chambers were maintained at 22.8 C (s = 0.6 ).
Alkalinity, pH, hardness, and acidity were measured at the beginning and the
end of each experiment (American Public Health Association 1971).
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LAKE
WATER I
NUTRIENT
SUPPLY
TO TEST CHAMBERS ,
TOXICANT
SYRINGE
Figure 1. Nutrient- and toxicant-adding diluter.
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Index mark
Float
Float in place
Figure 2., Duckweed testing chamber and float.
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The flow rate into the system was 1.2 1 of water (s = 0.13 1) delivered
every 15.6 min (s = 2.0); this was mixed with 2.5 ml (s = 0.15 ml) of nutrient
solution.
Aeration of the test chambers was not necessary. Lemna minor fronds have
adequate gas exchange through their dorsal surfaces.
Biological Methods
The clone that was used (Lemna minor L. #6) was originally collected in
Florida in the vicinity of Boca Raton some time before 1964. This clone has
been in culture at the Environmental Research Laboratory-Duluth since 1971.
Except where otherwise noted, colonies of two fronds each were used to
start a test. One colony was placed in each retaining ring of the float.
This resulted in seven colonies per jar and, since each concentration was
replicated four times, 28 colonies per test concentration.
To reduce possible effects of attached algal growth on surfaces in contact
with the test solution, on the 4th day of a 7-day test the colonies were
transferred into clean floats in clean battery jars. The water in the
replacement battery jars was lake water that had been allowed to equilibrate
to room temperature for 16-24 hrs. When copper toxicant was used, the water
in the jars was dosed with the appropriate concentrations of copper before
the transfer. Thus the duckweed was continuously exposed to the copper
concentrations. In the copper experiment the glassware cleaning procedure
included acid washing.
In- the course of experimentation two kinds of nutrients were used. The
first was a commercial soluble fertilizer (Garden Life, Science Products
Company, Chicago, Illinois 60614) with an analysis 10% nitrogen, 15% phosphorus,
and 14% potassium. The stock concentration was 1.24 mg/1. The second
nutrient was Hutner's Medium (Hutner 1953).
Stock cultures were maintained continuously under the same conditions as
the test organisms, lacking only the toxicant additions.
Growth rate (K) was calculated by the formula
Log1Q (Fd) - Log 1Q (Fo)
K = ~ ~
where Fo is the original frond number, Fd is the frond number on day d^, and
d is the number of days involved (Hillman 1961). When analysis of variance
or regression were used, individual frond counts were used, not chamber
averages.
Plant response for the first 2 or 3 days reflects a combination of
experimental and pre-experimental conditions; therefore, growth rates from
this period were not used (Fromm 1960). Because it is difficult to maintain
identical light intensities at the surface of the experimental containers
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and because the light output of fluorescent tubes changes with time, two
experiments were undertaken to determine whether the light variations over
the test bench correlated significantly with frond numbers at termination.
An experiment was undertaken to correlate the effects of the differing
flow rates into the separate containers with the frond numbers at termination.
A comparison was made between a series of static concentrations of a commercial
soluble fertilizer (Garden Life) and a continuously replaced solution of the
same material.
The purpose of another experiment was to determine when reduction of
growth due to crowding within the individual float chambers became significant.
Chambers were given six two-frond colonies each instead of one.
Because unequal amounts of damage may be caused in handling or in the
reduction of the colonies to two-frond units, minimally handled colonies
were compared with colonies that might be expected to show a maximum response
to handling. The unmodified colonies were simply transferred intact from
stock cultures to test chambers. The modified colonies were carefully broken
into two-frond units and the roots were excised.
The method developed here was tested with copper sulfate as the toxicant.
ED50 used here indicates a reduction of the production of new fronds by half;
this is derived from the difference between the final frond number and the
original frond number. Because copper at 31 ppb produced more growth than
the control Lake Superior water at 1 ppb, growth reduction was calculated
from this level as a starting point.
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SECTION 4
RESULTS
Effect of Light Variation
In two experiments no correlation was found between variations in light
intensity at frond level and frond numbers at termination. The mean of the
first experiment was 2,317 lux (s = 219) and of the second experiment, 2,749
lux (s = 221).
Effect of Flow-Rate Variation
A significant correlation (0.57) was found between flow rate and final
frond number in each test chamber (Figure 3).
Effect of Static vs. Flowing Conditions
The flowing concentrations of the soluble fertilizer at 1.24 mg/1
produced growth that was not significantly different from the static
concentration of the same fertilizer at 100 mg/1 (Figure 4).
Effect of Handling
Reduction of the colonies to two-frond, root-excised colonies did not
produce a significant reduction in growth rate.
Effect of Copper Sulfate
Linear regression was used for the data from 31 ppb to 210 ppb. The
7-day ED50 was 119 ppb copper, the upper confidence limit was 151, and the
lower confidence limit was 105 (Figure 5).
Effect of Crowding
A statistically significant reduction in growth was noted when the area
within the individual float chamber was reduced to 8.0 mm2 per frond. Three
days before this area was reached the available area was 12.5 mm2 per frond.
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50
0
&
E
rs
c
Q
30
j 1
Flow rate (ml/cycle)
75
.Figure 3. Correlation between flow rate in chambers and frond numbers at
end of 7 day test. Cycle time is 3.84 cycles per hour.
10
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Q>
E
12
10
o
Q.
I 8
S.
CD 6
73
O
O 2
il
N=77
N=84
N=84
* significantly different
from control
N=77
N=84
control
flow-through
0.001 g/l
static
1.0 g/l
static
O.I g/l
static
0.01 g/l
static
0.001 g/l
Figure 4. Growth differences between flowing and static conditions at various nutrient concentrations.
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o
e
"4
c
o
o
0)
0
ED50
10 20 40 100 200 300 500
Copper (ppb)
Figure 5. Growth responses to copper, as copper sulfate.
12
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SECTION 5
DISCUSSION
These experiments were undertaken in an attempt to develop a flow-
through testing procedure with duckweed as the test organism. The method
was produced by combining earlier work in this area with simplifications
derived from experiments. The goal was to create fast, simple, and inexpensive
techniques for evaluating stimulatory or inhibitory effects of various
pollutants. Where comparison is possible, the results produced here are
consistent with those reported earlier. However, in much of the research with
duckweed one or more of the important variables are poorly controlled.
Light output of the fluorescent tubes can vary over a fairly wide range,
both in space (across the table) and time (between experiments), so it
becomes necessary to monitor these changes and periodically determine whether
there are significant correlations between light intensities and growth rates
over the intensity range utilized. The combination of fluorescent tubes used
here (50:50 Gro Lux Wide Spectrum and Duro Test Vita Light) is standard
practice at the Environmental Research Laboratory-Duluth. The industry-derived
Color Rendering Index (CRI) for each of these is 55 and 92, respectively.
Daylight is assigned a CRI of 100. If a better approximation of daylight is
needed, only tubes having a CRI of greater than 90 should be used.
Flow rates vary among chambers, and growth rates can vary significantly
with them. This flow variation should be kept to a minimum, and the
correlation between flow rate and growth rate should be determined periodically.
Flowing conditions require far less in the way of nutrient concentration.
This can have the effect of reducing the amount of interaction between the
necessary nutrients and the material being tested.
The reduction of the colonies to a standardized colony (two fronds, roots
exicised) causes no reduction in growth rate. It then is reasonable to start
with such a standard colony for reasons of uniformity and because the
statistical treatments are somewhat simplified.
In the test of copper sulfate toxicity, which served here only as a
test of the procedure, it should be noted that the 7-day ED50 of 119 ppb
copper indicates only part of the copper toxicity problem with duckweed.
Copper is one of several metals that bioaccumulate in plants so that subsequent
complications are to be expected.
13
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The effects of crowding were apparent at 8.0 mm2 per frond, but because
the response of duckweed takes an area of 2-3 days to appear, 12.5 mm2, the
area per frond 3 days earlier, is taken as the upper limit of allowable
crowding.
This system or a similar one may be adapted to study the effects of air
pollution on plants.
14
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RECOMMENDED METHODS
The following recommended experimental conditions and procedures are
based on the results of the experiments described.
1) Initial conditions and measurements:
a) Determine whether the variations in growth are correlated with
variations in light intensity or flow rate. In any case, light intensity
and flow rate should be as constant as possible. Because changes in light
intensity and flow rate are relatively slow, measurements may be made between.
not during, experiments.
b) For starting the test and replacing the glassware, use water
of the same temperature and the same concentration of toxicants as that from
which the plants are being transferred.
c) The stock cultures of experimental plants should be maintained
in a situation as close to the experimental conditions as possible. A two-frond,
root-excised standard colony reduces transfer of epiphytic algae and makes
growth rates of colonies directly comparable.
d) A proportional diluter is not necessarily the only way to provide
the toxicant solutions, but any system used should have fail-safe operation,
so that if water flow stops, toxicant flow stops as well.
2) Steps should be taken to reduce algal growth wherever possible. The
experiments should be designed so that individual colonies can be kept track
of separately. Lighting should be continuous.
3) Light intensity should be around 2,500 lux with a standard deviation of
250 or less.
4) Temperatures in the test chambers should be held close to some point
between 22°and 25°C with a standard deviation of 0.6 C or less. Infra-red
radiation from the fluorescent tubes keeps the chambers slightly above room
temperature.
5) For most purposes a 7-day exposure is adequate, but the first 3 days of
data are not useful because the plants are still adjusting to their new
environment. The glassware may be changed to reduce the influence of epiphytic
organisms on or about the fourth day of the test.
15
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6) Toxicant concentrations as well as pH, hardness, and alkalinity should
be measured at least three times in the course of a 7-day experiment. The
most convenient times are when the fronds are added, when the chambers are
changed to reduce periphyton growth, and when the experiment is terminated.
7) Aeration of test chambers is not necessary.
8) Fronds should be counted at the same time every day.
9) If a notched battery jar chamber is used as described here, stainless
steel screen over the notch is not essential. Any reasonably inert material
will do to hold back fronds that might escape their enclosures. Whatever
form the chambers take, the table they rest on should be marked so that they
can be placed as consistently as possible.
16
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REFERENCES
American Public Health Association. 1971. Standard methods for the examination
of water and wastewater. 13th ed. New York, N.Y.
Ashby, Eric, B. D. Bolas, and F. T. Henderson. 1928. Interaction of factors
in the growth of Lemna; I Methods and Technique. Ann. Bot. 42:771-782.
Ashby, Eric, and T. A. Oxley. 1935. The interaction of factors in the growth
of Lemna: VI An analysis of the influence of light intensity and
temperature on the assimilation rate and the rate of frond multiplication.
Ann. Bot. 49:309-336.
Blackman, G. E. 1952. Studies in the principles of phytotoxicity. I The
assessment of relative toxicity. J. Exp. Bot. 3:1-27.
Clark, N. A. 1925. The rate of reproduction of Lemna major as a function
of intensity and duration of light. J. Phys. Chem. 29:935-941.
DeFoe, D. L. 1975. Multichannel Toxicant Injection System for Flow-Through
Bioassays. J. Fish. Res. Board Can. 32(4):544-546.
Eichorn, M., and H. Augsten. 1969. Kontinuierliche Kultivierung von Wolffia
arrhiza (L.) Wimm. im Chemostaten. (Continuous cultivation of Wolffia
arrhiza (L.) Wimm. in a chemostat. Flora, Abt. A, 160:576-580.
Erismann, K. H., and A. Finger. 1968. Lemnaceen in kontinuierlicher Kultur.
(Lemnaceae in continuous culture). Ber. Schweiz. Bot. Ges. 78:5-15.
Feder, W. A., and F. Sullivan. 1969. Ozone: depression of frond multiplication
and floral production in duckweed. Science 156:1373-1374.
Fekete, A., D. N. Riemer, and H. L. Motto. 1976. Bioassay using common
duckweed to evaluate the release of available phosphorus from pond
sediments. J. Aquatic Plant Manage. 14:19-25.
Fromm, F. 1946. El Empleo de Lemna minor L. en Ensayos Rapidos de Toxicidad.
Cienciu. 7:214-218.
Fromm, F. 1951. A quantitive evaluation of the Lemna test for herbicides.
Bot. Gaz. 113:86-90.
Fromm, F. 1960 A modification of the Lemna test for phytotoxicity. J. Agr.
Univ. Puerto Rico 46:93-102.
17
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Hero of Alexandria, Ca. 62 AD. Pneumatics, Section 12. (ed. Woodcroft, The
pneumatics of Hero of Alexandria. Taylor, Walton and Mabery. London
1851).
Hillman, W. 1961. The Lenaceae, or duckweeds: a review of the descriptive
and experimental literature. Bot. Rev. 27:221-287.
Hutner, S. H. 1953. Comparative physiology of heterotrophic growth in plants,
in W. E. Loomis [ed.] Growth and differentiation in plants. Iowa State
College Press, Ames, la.
Landolt, E. 1957. Physiologische and okologische untersuchungen an
Lemnaceen. (Physiological and ecological investigations of the Lemnaceae)
Ber. Schweiz. Bot. Ges. 67:272-410.
Mandiola, N. B. 1919. Variation and selection within clonal lines of Lemna
minor. Genetics 41:151-182.
Mitchell, J. W., G. A. Livingston, and P. C. Marth. 1958. Test methods with
plant regulating chemicals. U.S. Dep. Agr., Agr. Handb. 126. pp. 34-36.
MDunt, D. I., and W. A. Brungs. 1967. A simplified dosing apparatus for fish
toxicology studies. Water Res. 1:21-29.
Muenscher, W. C. 1944. Aquatic plants of the United States. Cornell
University Press, Ithaca, N.Y. 374 p.
Parker, C. 1965. A rapid bioassay method for the detection of herbicides
which inhibit photosynthesis. Weed Res. 5:181-184.
Scharer, M., C. Brunold, and K. H. Erismann. 1975. Hemmung der Sulfataufnahme
durch Lemna minor L. durch SC-2 in subletalen Konzentrationen. (Inhibition
of sulfate uptake by Lemna minor L. during aeration with sublethal
concentrations of S02.) Specialia:1414-1415.
Sculthorpe, C. D. 1967. The biology of aquatic vascular plants. Arnold,
London. 610 p.
Strasser, R. J. 1971. Eine einefache Anlage zur kontinuierlichen Kultivierung
von Lemnaceen mit automatischer probeentnahme. (Simple apparatus for
continuous cultivation of Lemnaceae with automatic sample collection.)
Photosynthetica 5:76-78.
Todd, G. W., J. T. Middleton, and R. F. Brewer. 1956. Effects of air
pollutants. California Agr. (July): 7, 8, 14.
18
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BIBLIOGRAPHY
Lemke, A. E., W. A. Brungs, and B. J. Halligan. Manual for Construction and
Operation of Toxicity Testing Proportional Diluters. U.S. Environmental
Protection Agency, Environmental Research Laboratory-Duluth, Ecological
Research Series (in press).
19
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/3-77-108
2.
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
A FLOW-THROUGH TESTING PROCEDURE WITH DUCKWEED
(LEMNA MINOR L.)
5. REPORT DATE
September 1977 issuing date
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
Charles T. Walbridge
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Environmental Research Laboratory - Duluth, MN
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
10. PROGRAM ELEMENT NO.
1BA608
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
Same as above.
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA/600/03
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Lemna minor is one of the smallest flowering plants. Because of its floating
habit, ease of culture, and small size it is well adapted for laboratory investiga-
tions. Procedures for flow-through tests were developed. Testing procedures were
developed with this apparatus. By using the techniques described here, the effects
of nutrients or toxicants, singly or in combination, can be determined in several
concentrations with several replicates of each. Responses which can be measured
include changes in growth rate, changes in death rate, changes in timing of division
of colonies, color changes, changes in variability, and modification of the flowering
response. Emphasis here is on changes in growth rate determined either by daily
frond counts or by final frond numbers.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TEFIMS
c. COS AT I Field/Group
Bioassay
Aquatic Plants
Plant nutrition
Growth regulators
Toxicity
Pollution
Botany
Plant regulators
Lemna minor L.
Plant responses
Laboratory plant
Proportional diluter
Toxicants
Nutrients
Pollutants
06 F
18. DISTRIBUTION STATEMEN1
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF P,
26
20. SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
•ft U.S. GOVERNMENT PRINTING OFFICE: 1977-757-056/6547
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